Oral cancer is a major health problem in the United States; it is a disease of high mortality and severe morbidity. While risk factors include alcohol and tobacco usage, some oro-pharyngeal tumors develop without a defined pattern of alcohol or tobacco abuse. In fact, the increased use of alcohol based mouth washes in the United States may be a risk factor. Although it has been reported that consumption of fresh fruits and vegetables is associated with protective effects on the development of oro-pharyngeal tumors in high risk groups, identification of specific nutritional components such as one specific antioxidant that protects against the development of this type of tumor has not been successful. The inability to isolate a protective effect of a specific vitamin (e.g., beta-carotene) might be secondary to synergistic protective effects of multiple dietary components or to inadequate previous study of methyl supplying components such as folate or vitamin B-12. Localized folate and vitamin B-12 deficiencies may be especially relevant to the development of these tumors since levels of active folate and vitamin B-12 in tissues may be decreased by exposure to components of tobacco or alcohol usage. We hypothesize that the normal proliferative homeostasis of basal cells of the oral epithelium is dysregulated by localized deficiencies of folate and vitamin B-12 and that the resulting abnormality in basal cell proliferation is exacerbated by exposure to alcohol and tobacco products. Ultimately, increased cellular proliferation increases immature cells at intermediate and higher layers (i.e., above the basal cell layer) in the oral epithelium and leads to the development of histologically recognizable pre-malignant characteristics in the epithelium (e.g., dysplasia). These changes can be identified by decreased regional vitamin levels and by changes in the expression of intermediate endpoint biomarkers (IEBs) in the proliferating or dysplastic epithelium. We will select tissues for evaluation based on standard epidemiological methods in order to validate our hypothesis. We will evaluate the relation of folate and vitamin B-12 levels geographically within the epithelium and their correlation with the development of neoplastic changes and with expression of the following IEBs: TGFalpha, EGF-R, EGF, p185/crbB-2, TGF-beta, fatty acid synthetase, markers of proliferation and p53. This proposal is strengthened by nesting state-of- the-art biochemical, molecular, and pathological assessments of normal, pre-malignant and squamous cell cancers within a sound epidemiological investigation of well characterized patients.